Limited solvation of an electron donating tryptophan stabilizes a photoinduced charge-separated state in plant (6–4) photolyase

(6–4) Photolyases ((6–4) PLs) are ubiquitous photoenzymes that use the energy of sunlight to catalyze the repair of carcinogenic UV-induced DNA lesions, pyrimidine(6–4)pyrimidone photoproducts. To repair DNA, (6–4) PLs must first undergo so-called photoactivation, in which their excited flavin adenine dinucleotide (FAD) cofactor is reduced in one or two steps to catalytically active FADH− via a chain of three or four conserved tryptophan residues, transiently forming FAD•−/FADH− ⋯ TrpH•+ pairs separated by distances of 15 to 20 Å. Photolyases and related photoreceptors cryptochromes use a plethora of tricks to prevent charge recombination of photoinduced donor–acceptor pairs, such as chain branching and elongation, rapid deprotonation of TrpH•+ or protonation of FAD•−. Here, we address Arabidopsis thaliana (6–4) PL (At64) photoactivation by combining molecular biology, in vivo survival assays, static and time-resolved spectroscopy and computational methods. We conclude that At64 photoactivation is astonishingly efficient compared to related proteins—due to two factors: exceptionally low losses of photoinduced radical pairs through ultrafast recombination and prevention of solvent access to the terminal Trp3H•+, which significantly extends its lifetime. We propose that a highly conserved histidine residue adjacent to the 3rd Trp plays a key role in Trp3H•+ stabilization.


Multiple sequence alignment
For assignment of plant and animal (6-4) PL orthologues, we performed blastp 1 searches using the At64 and Xl64 amino acid sequences as representative plant and animal  photolyase sequences against the refseq_protein database with an E-value threshold of 10 -150 . The resulting sequences (202 sequences for plants and 1471 sequences for animals) were aligned with COBALT 2 .
From the multiple sequence alignments, the WebLogo 3 was created with the version 3.7.4. A480 of 0.0433 converts to a transmittance T480 of 90.51%, i.e. 9.49% of the excitation light was absorbed by the actinometer over the 2 mm path. The flashes induced an absorption change of ΔA457 = 0.0465 (recorded over 1 cm optical path; 64 signals were averaged). Assuming that the Δε457 value for the formation of the 3 MLCT (metal-to-ligand charge transfer state formed with a ~100% quantum yield) is close to the -1.1 × 10 4 M -1 cm -1 as estimated for 450 nm 5 (which is reasonable given the shape of the difference spectrum 6 ), one obtains a concentration of the excited [Ru(bpy)3] 2+ complexes / formed 3 MLCT states of ~4.23 µM. In the excited volume of 40 µL, this concentration corresponds to ~1.69 × 10 -10 mol of absorbed photons (out of the total ~1.78 × 10 -9 mol, given that only 9.49% photons were absorbed). Since 480 nm photons have an energy of 249 220 J/mol, the energy absorbed by the [Ru(bpy)3]Cl2 actinometer was ~42 µJ. Considering that the window through which the sample was excited had a surface of 0.2 cm 2 (0.2 × 1.0 cm) and that only 9.49% of the excitation light was absorbed, one obtains an excitation energy (per pulse and per cm 2 ) of 2.23 mJ.

Estimation of the excitation energy and of the quantum yield of 'stable' FAD
The ~125 µM At64-WT sample had an absorbance A480 of 1.0334 over the 1 cm path and 0.2067 over the 2 mm path at the excitation wavelength. A480 of 0.2067 converts to a transmittance (TrpH does not absorb at 457 nm). The observed initial amplitude of the At64-WT signal at 457 nm ΔA457 (t→0) = -0.065 hence corresponds to ~14.1 µM FAD •-TrpH •+ pairs, which is ~83% of the maximum of pairs that could have hypothetically been formed by the excitation flash-.
Even though we do not know some of the ε values precisely because the absorption spectra of the FAD •and TrpH •+ radicals can (and do) slightly vary from protein to protein, the quantum yields of most of the other PCSf proteins mentioned in the main text were determined by the same method (and essentially confirmed by later ultrafast experiments), so it is safe to say that the losses due to ultrafast recombination of the FAD •-Trp1H •+ and FAD •-Trp2H •+ radical pairs are indeed significantly lower in At64 than in the other studied PCSf proteins and amount to ~20%.
When the vertical axes are scaled to reflect the difference in protein (FAD) concentrations ( Fig. 6 of the main text), the initial amplitudes of the signals obtained for the H382S mutant are practically the same as for the WT protein, indicating that the quantum yield of the 'stable' FAD •-TrpH •+ pairs (and the losses through their ultrafast recombination) are very similar in both proteins.
Using the same method of calculation as for the WT protein, we estimate that the excitation of the ~95 µM At64-H382S, with A480 = 0.1545 over the 2 mm path and ΔA457 (t→0) = -0.048 over the 1 cm path, yielded ~10.4 µM FAD •-TrpH •+ pairs, which is ~78% of the maximum ~13.3 µM of pairs that would have been formed in a hypothetical case of a 100% quantum yield.

Molecular dynamics simulations for solvation analyses
Molecular dynamics (MD) simulations for solvation analyses were conducted with the AMBER 16 program package 7 , as reported previously for the simulation of At64-WT 8 . The initial structures of H382D, H382N, H382S, H382V, and H382Y mutants of At64 were generated with SWISS-MODEL 9 using the At64-WT crystal structure (PDB entry: 3FY4) as the template structure.
We applied the Amber force field 14SB 10 and the previously prepared Amber force field 8 for the proteins and FADH − , respectively. We solvated the proteins with TIP3P water model 11 in the simulation boxes with a margin of 12 Å from the proteins to the box boundaries and neutralized the system by adding some counter ions (Cl -). In the following MD simulations, the SHAKE algorithm was used for the constraints 12 , the periodic boundary condition with the particle mesh Ewald method was applied 13 , and the simulation time step was set to 2 fs. The energy minimization for each system with the Sander module was performed for 5,000 steps with 10 kcal mol -1 Å -2 of restrictions on heavy atoms, and for 10,000 steps without any restrictions. After the system temperature was increased from 0 to 300 K for 100 ps with the NVT ensemble (T = 300 K), the system was equilibrated for 1 ns with the NPT ensemble (P = 1 atm and T = 300 K). Then, 200 ns MD simulation in the NPT ensemble (P = 1 atm and T = 300 K) was performed. The last 100 ns of the trajectory for each system was recorded every 20 ps and subjected to our analyses. For At64-WT, the last 100 ns of the trajectory previously simulated in the same way 8 was used for our analyses.

Molecular dynamics simulations for estimation of electron transfer parameters
We estimated electron transfer parameters in the photoreduction of the fully oxidized FAD (FADox) in At64-WT and H382S mutant through the Trp-triad chain by using classical MD simulations. The force field parameters for FAD were prepared by using the Antechamber module in we imposed the constraint condition on the RESP charges of FAD •identical to those of FADox except for the isoalloxazine-ring part. The atomistic coordinates of At64-WT and its H382S mutant were prepared using At64-WT crystal structure (PDB entry: 3FY4) like in the previous section. We set the force-field parameters of protein and the crystal water molecules to ff14SB 10 and TIP3P 11 , respectively. In addition, the partial atomic charges of the oxidized Trp for Trp2H •+ and Trp3H •+ were evaluated by the RESP fitting scheme to the B3LYP/cc-pVDZ results, where we imposed the constraint condition on the RESP charges identical to those of the neutral Trp except for the side chain. We solved the constructed models of the At64-WT and H382S mutant in a truncated octahedron box of TIP3P water molecules having an edge distance of 12.0 Å from the protein. We added six counter ions (Cl -) for electroneutrality.
First, we set the charge states of FAD and the Trp-triad to FADox and neutral Trp, respectively, for both the simulation systems of the At64-WT and H382S mutant. We performed the energy minimization (15,000 steps) for each system with the PMEMD module in the Amber18 suite; we imposed 50 kcal mol -1 Å -2 of harmonic restrictions on all the heavy atoms during the first 5,000 steps.
Then, we did the following MD simulations by using GPU-accelerated PMEMD module [19][20][21] in the Amber18 suite with the SHAKE algorithm, the particle mesh Ewald method, a non-bonding cut-off distance of 10 Å, and a time step of 1 fs. We slowly heated both systems up to 300 K within 300 ps and kept them within the following 200 ps in the NVT ensemble, while maintaining 50 kcal mol -1 Å -2 of restrictions on the heavy atoms of protein and FAD. During the first equilibration phase, we did five-successive 200 ps MD simulations in the NPT ensemble by gradually decreasing the harmonic restrictions (50, 20, 10, 5, and 1 kcal mol -1 Å -2 ). During the second equilibration phase, we did the additional 100 ns MD simulations in the NPT ensemble without any restrictions. At the beginning of the third equilibration phase, we changed the charge states of FAD and the Trp-triad; one set was FAD •and Trp2H •+ (Trp1H and Trp3H are neutral) and the other one FAD •and Trp3H •+ (Trp1H and Trp2H are neutral). The former and latter charge states correspond to the initial and final states of the electron-transfer from Trp3H to Trp2H •+ (or the hole transfer from Trp2H •+ to Trp3H). During the third equilibration phase, we did 100 ns MD simulations on the initial and final ET states of the At64-WT and its H382S mutant systems in the NPT ensemble. Finally, during the subsequent 100 ns production MD simulations in the NPT ensemble, we collected the snapshots every 10 ps for the following free energy difference (ΔG) and reorganization energy (λ) calculations.
Within the linear response approximation, one can evaluate the ΔG and λ in the Marcus rate formula from the ensemble of the vertical energy differences between the initial ( ) and final ( ) ET states, !" , as follows: 20,21 Here, 〈 〉 ! and 〈 〉 " represent the thermal averages over the initial and final ET states, respectively. Since